Vinculin stabilizes nascent adhesions and establishes lamellipodium-lamella border in migrating cells Ingo Thievessen, R. Ross The actin cytoskeleton at the leading edge of migrating cells consists of two actin networks, the lamellipodium (LP), characterized by fast polymerization-driven retrograde actin flow and the lamellum (LM) with slow myosin II (myoII) mediated actin flow. The engagement of LP actin to the ECM via nascent integrin-mediated focal adhesions (FA) establishes the flow velocity gradient between LP and LM. Nascent adhesions then elongate and mature via myoII LM actin flow. How integrins are connected to the retrograde actin flow is not known. Using primary murine embryonic fibroblasts (MEF) deficient for the vinculin gene (Vcl), we sought to test the hypothesis that vinculin mediates the coupling of actin retrograde flow to the ECM in FA . Single Vcl-/- MEF migrated faster than control (Vclfl/fl) MEF and displayed impaired anisotropic spreading. To determine if LP/LM organization were effected by vinculin deletion, we analyzed distributions of phospho-myosin light chain (pMLC), cortactin, and paxillin. This revealed a shift in pMLC distribution towards the cell edge, reduced LP paxillin intensity, and broadening of the contactin band at the leading edge. This suggests loss of delineation between the LP and LM. To test this, we performed spinning disc confocal (SDC) microscopy of MEF containing fluorescent paxillin and actin. This revealed reduction in the rate of formation of short-lived, diffraction limited FA in the LP of Vcl-/- MEF, indicating an impaired stabilization of nascent LP FA. Kymograph analyses of high resolution DIC and quantitative fluorescent speckle SDC microscopy of actin indicate the lack of two distinct velocity zones of retrograde f-actin flow near the leading edge and an increased retrograde flow velocity in the LM region of Vcl-/- MEF. We suggest that vinculin stabilizes nascent FA by coupling to lamellipodial actin flow, thus establishing the flow velocity gradient between LP and LM and promoting the maturation of nascent adhesions. This implicates vinculin as an essential component in linking the dynamic actin cytoskeleton to the ECM during cell migration. This work was done in collaboration with Bob Ross, and has been presented at Cell Biology Society meeting and Gordon Conference and a manuscript describing these results has been submitted for publication. Project 2: Nanoscale architecture of integrin-based cell adhesions Lindsay Case Specific protein-interactions regulate the three-dimensional nanoscale organization of vinculin within focal adhesions L.B. Case, G. Shtengel, H.F. Hess, C.M. Waterman Vinculin is a ubiquitously expressed protein that localizes to FAs in a myosin II-dependent manner, and is important for regulating FA functions including adhesion strength, actin dynamics, and signaling. Vinculin has at least 8 putative binding partners at FAs including the FA. Thus, vinculin may regulate specific FA functions via interaction with particular binding partners. Interferometricphotoactivated localization microscopy (iPALM) is a superresolution light microscopy technique that allows for 20nm lateral resolution and 10nm axial resolution. We previously used iPALM to show that FAs have a conserved stratified structure. We are currently using iPALM to test the hypothesis that the vertical position of vinculin within the FA is dictated by specific protein-protein interactions that modulate its function in reinforcing FA strength, signaling, and regulating actin dynamics. We have focussed first onvinculinspaxillin and actin binding interactions.Vinculin interaction with paxillin requires FAK-mediated phosphorylation ofpaxillin, and the vinculin-actin interaction requires vinculin activation. We showed previously that paxillin is located 5nm axially from the plasma membrane within FAs, while actin is located 30-40 nm higher, suggesting that two spatially and functionally distinct populations of vinculinmay exist in FAs.Here we found that phosphomimic mutations in paxillinpromotevinculin accumulation near the plasma membrane, while FAK inhibition or non-phosphorylatablepaxillinmutants shiftvinculin localization 30-40 nm higher within FA. We conclude that vinculin interacts with phospho-paxillinnear the plasma membranewithin FA.Furthermore, we show that iPALM allows high-resolution measurements of changes in vinculins 3D localization within the focal adhesion structure, suggesting that iPALM is a valid technique to understand the contribution of vinculin binding interactions to vinculinsnanoscaleorganization and function within FA.We are currently mapping the 3D nanoscale organization of various vinculin mutants and correlating this nanoscale organization with vinculins FA function. Project 3: Formin INF2 localizes to Focal Adhesions and is Critical for Focal Adhesion Function Colleen Skau Focal adhesions (FAs) are mechanosensitive complexes that form the physical and signaling link between the extracellular matrix and the actin cytoskeleton and are critical to driving cell migration. During migration, FAs form at the cell leading edge then undergo force-dependent maturation during which they elaborate a dorsal stress fiber to allow integration of force transmission across the cell. The formin family of actin nucleators are thought to be critical to FA maturation and formation of dorsal stress fibers. A proteomic analysis of FAs from our lab revealed two formin family members as FA components. We investigated the role of one of these, the formin INF2, in assembly and maturation of FAs and their associated stress fibers in mouse embryonic fibroblasts. We found that green fluorescent protein-tagged INF2 associated with FAs, partially co-localizing with known adhesion proteins including paxillin, VASP, and vinculin. INF2 localized to the rear of paxillin-marked FAs, partially co-localizing with the growing stress fiber. We utilized siRNA-mediated knockdown of INF2 to determine its role in FA and stress fiber formation and dynamics. Knockdown of INF2 had no effect on localization of paxillin, vinculin and VASP to FA, however had major effects on FA morphology. Compared to control, in cells with reduced INF2, FAs were smaller, more numerous, and failed to elongate, indicative of a possible maturation defect. Examination of the actin cytoskeleton in cells with reduced INF2 showed that although both stress fibers and dorsal arcs were present, INF2 knockdown cells contained fewer dorsal stress fibers, and actin bundles in both stress fibers and arcs were wavy and disorganized. To determine the requirement for INF2 in cell migration, we tracked migration of individual cells. INF2 knockdown cells also exhibited migration defects migrate displaying an abnormal jumping motility. These results suggest that INF2 participates in coordinating adhesion and contraction during cell migration by integrating FA maturation and stress fiber formation. Project 4: The role of actin polymerization in regulating endothelial cell barrier function Case, LB, and Waterman CM Endothelial cells form a selectively permeable barrier in blood vessels and arteries. Endothelial cells are exposed to a variety of extracellular stimuli including shear force, pulsatile stretching, soluble factors such as growth factors and chemokines, as well asleukocyte adhesion and transmigration. Since the endothelial cytoskeleton is known to play a role in regulating vascular permeability, we sought to test the hypothesis that different stimuli may compromise barrier function via specific effects on the actin cytoskeleton of endothelial cells. In this study, we examine how the endothelial actin cytoskeleton and cell-ECM adhesions change in res